Apoptotic cell-mediated transfection of mammalian cells with interfering rna

Abstract

Mammalian host cells for use in a cell-mediated tranfection process, which contain an RNAi molecule and an expression vector for a pro-apoptotic protein. The method includes inducing apoptotic cell (AC) death in mammalian cells that contain an RNAi molecule capable of downregulating a chosen target gene. Living cells expressing the target gene are then exposed to the ACs. The ACs are processed by the living cells, and the RNAi molecule in the ACs downregulates the expression of the target gene in living cells.

1 - 23 . (canceled) 
     
     
         24 . An isolated pre-apoptotic mammalian cell, comprising:
 (a) an RNAi molecule capable of downregulating a target gene; and   (b) an expression vector capable of expressing a pro-apoptotic protein.   
     
     
         25 . The mammalian cell of  claim 24 , wherein said RNAi molecule is a short interfering RNA (siRNA) or a short hairpin RNA (shRNA). 
     
     
         26 . The mammalian cell of  claim 25 , wherein said siRNA comprises a short double-stranded RNA (dsRNA) region of about 19-23 base pairs, and each strand of the siRNA further comprises a single-stranded overhang of about two nucleotides at its 5′ or 3′ end. 
     
     
         27 . The mammalian cell of  claim 24 , wherein said RNAi molecule comprises a short double-stranded RNA (dsRNA) region of about 19-27 base pairs. 
     
     
         28 . The mammalian cell of  claim 24 , wherein said RNAi molecule is encoded by a vector capable of expressing a short hairpin RNA (shRNA) or a short interfereing RNA (siRNA). 
     
     
         29 . The mammalian cell of  claim 28 , wherein said vector comprises at least one RNA polymerase III promoter that controls the transcription of said RNAi molecule. 
     
     
         30 . The mammalian cell of  claim 24 , wherein said pro-apoptotic protein is BAX. 
     
     
         31 . The mammalian cell of  claim 24 , wherein said mammalian cell is converted to an apop o ic cell upon expression of said pro-apoptotic protein. 
     
     
         32 . The mammalian cell of  claim 24 , further comprising an antigen. 
     
     
         33 . The mammalian cell of  claim 32 , wherein said antigen is an autoantigen. 
     
     
         34 . A method of treating an autoimmune disease, comprising: administering to a subject in need thereof a therapeutically effective amount of the mammalian cell of  claim 33 . 
     
     
         35 . The mammalian cell of  claim 32 , wherein said antigen is a donor antigen. 
     
     
         36 . A method of treating rejection of a transplanted donor organ, comprising: administering to a subject in need thereof a therapeutically effective amount of the mammalian cell of  claim 35 . 
     
     
         37 . The mammalian cell of  claim 24 , wherein said target gene is CD40. 
     
     
         38 . A method of inducing immune-tolerance, comprising: administering to a subject in need thereof a therapeutically effective amount of the mammalian cell of  claim 37 .